Rescue and repair of stalled ribosome damaged by ribosome-specific ribotoxins

项目摘要/摘要 Colicin E3是50年前第一个被鉴定的核毒素。它只做了一次但精确的切割 细菌核糖体解码中心的16S rRNA，导致核糖体和最终细胞停滞 死亡。在过去的半个世纪里，尚不清楚是否存在一种能够 逆转结肠素E3对核糖体的破坏，使细胞得以存活。采用以下方法 生物信息学、生物化学、结构生物学和微生物学，我们发现了两种细菌-- 组份RtcB和PrfH，作为Colicin E3的解毒剂。具体来说，细菌PrfH识别 核糖体受损和停滞，并执行核糖体抢救。然后是RtcB修复 30S核糖体亚基受损。上面描述的连续事件有大量的支持 来自我们的体外和体内研究的初步数据。在此应用程序中，我们计划显著扩展 我们系统地研究了细菌核糖体的挽救和修复 解码中心的具体损坏有以下三个主要目标：(1)我们将提供对 细菌PrfH在体外识别和挽救受损和停滞的70s核糖体；(2)我们将 细菌RtcB的体外生化和结构特征，重点是PrfH偶联的RtcB 修复受损的30S核糖体亚基；以及(3)我们将阐明RtcB-的体内生物学功能。 PrfH使用我们开发的体内减弱的RNA损伤系统。

Project Summary/Abstract Colicin E3 was the first ribotoxin to be characterized 50 years ago. It makes a single but precise cut of 16S rRNA in the decoding center of bacterial ribosome, resulting in stalled ribosome and eventual cell death. Over the last half of a century, it is unclear whether there exists a biological system that is able to reverse the ribosomal damage by colicin E3 to allow cell to survive. Employing approaches of bioinformatics, biochemistry, structural biology, and microbiology, we have uncovered a bacterial two- component system, RtcB and PrfH, as the antidote of colicin E3. Specifically, bacterial PrfH recognizes the damaged and stalled ribosome and performs ribosomal rescue. This is followed by RtcB repairing the damaged 30S ribosomal subunit. The sequential events described above are supported by abundant preliminary data from both our in vitro and in vivo studies. In this application, we plan to significantly expand our preliminary studies to systematically characterize the rescue and repair of bacterial ribosome with specific damage in the decoding center with the following three main aims: (1) We will provide insight into bacterial PrfH recognizing and rescuing the damaged and stalled 70S ribosome in vitro; (2) We will biochemically and structurally characterize bacterial RtcB in vitro, with the emphasis of PrfH-coupled RtcB repairing the damaged 30S ribosomal subunit; and (3) We will elucidate in vivo biological functions of RtcB- PrfH using an in vivo attenuated RNA damage system we have developed.